1. Field of the Invention
The present invention relates generally to devices and methods for screening, selecting, and validating small molecule and biologic drug candidates in solution and, more particularly, to spatially distributed identification tags that facilitate the direct quantitative, semi-quantitative, or qualitative assay of proteins, genes and their biologic products (carbohydrates, lipids or other cellular components).
2. Related Art
All patents and publications cited throughout the specification are hereby incorporated by reference into this specification in their entirety in order to more fully describe the state of the art to which this invention pertains.
The mapping of the human genome, currently believed to comprise of some thirty thousand genes, has led to an exponential growth in data available to pharmaceutical companies. The linkage between specific genes and disease processes, namely functional genomics, will, it is believed, provide a means of better screening small molecule libraries against druggable genes (genes that are believed to be functionally related to a specific disease). Small molecules will be selected on the basis of their ability to influence the expression profile of the messenger RNA (mRNA) of such genes. There are, however, limitations to such selection strategies. Firstly, the strategy assumes that all gene products are known and can be assayed; secondly, the strategy assumes a one to one relationship between gene and protein. The occurrence of gene-splice variants in all but infectious diseases and prokaryotic species belies the former assumption, whilst post-translational modification (e.g., glycosylation, phosphorylation, acetylation) of proteins as well as environmental parameters obviate the latter assumption. As a consequence of these limitations, proteomics, whose aim is the mapping of proteins, protein-protein interactions, and their metabolic, catabolic, and anabolic pathways, has grown in significance as a means of facilitating the selection of small molecules, and biologics (e.g., protein therapeutics, monoclonal antibodies, vaccines, therapeutic serum or gene transfer products), against a growing number of established, and yet to be established, protein targets. The emerging field of proteomics is estimated to yield in excess of 10,000 proteins during this decade. Identification and validation of these potential targets will require substantial equipment, supplies for testing in-vitro with cell based assays, in-vivo with animal models and eventually with human clinical trials in order to ensure drug discovery and subsequent development.
Such testing requires small molecules or biologics to be screened against samples of tissue and physiological fluids, comprising the protein, genes, or other biological (carbohydrate, lipid) targets of interest. Such samples can be costly and difficult to access, often requiring a priori confirmation that the testing to be conducted is therapeutically relevant and justified. A blood sample likely contains greater than 10,000 protein targets, yet existing instrumentation and assay methodology limits the number that can be realistically used as targets to a far smaller figure. A pharmaceutical company will typically screen all of its small molecule against less than one percent of this figure, necessarily eliminating many potential drug targets, and possible drug candidates.
This strategy requires sophisticated instrumentation that can purify drug leads, screen the vast number of leads with their protein targets, and analyze and interpret the results. The primary methods used typically involve each or a combination of the following: liquid chromatography (LC) an expensive but well established method primarily for the distillation of drug leads, 2d (Isoelectric Focusing) and 1d-gel electrophoresis (SDS-PAGE), yeast 2-hybrid systems, mass spectrometry (MS), and various types of immunoassay. All of these methods involve well established but costly instrumentation, requiring specialist expertise for their operation and interpretation of data. These methods do not provide, either individually or collectively, a method of rapidly screening a massive number of small molecules or biologics against an equally massive number of protein targets. This is, in part, due to the fact that not all protein targets are known. 2d-gel electrophoresis is the primary method for the mapping out of proteins (estimated at between 60,000 to 150,000 per mammal) but has resolution limits imposed by the distance between spots and the protein loading per spot, thus significantly limiting the technique to high abundance proteins. This is a severe limitation given that many low abundance proteins are believed to play key roles in cellular signaling and disease pathways, and that protein activity provides more therapeutically valuable information than protein abundance. Moreover proteins often do not act alone. An increasing effort is being spent on examining how proteins interact, not only with other proteins but also with nucleic acid, small molecules and ligands. A current popular method is to use antibodies as capture molecules to trap interacting proteins. The immunoprecipitate is then run out on a 1-D gel, digested and analyzed by tandem MS to determine the identity of the interacting partners. Yeast 2-hybrid systems are powerful tools for the identification of protein-protein and protein-DNA interactions, although they are hampered by high rates of false positives, a poor ability to identify weak interactions, a relatively low throughput and are not suited generally to the study of protein-ligand interactions. MS requires “clean” samples and is not good at analyzing protein complexes. Inmunoassays provide a means of determining the kinetics and cross-reactivities associated with the binding of drug targets to drug compounds. They are usually conducted in microtiter plates of either 96-well or 384-well format. However, the serial nature of this process combined with the requirement for washing, incubation, and heavy reagent consumption, mean that this is a costly and time-consuming process.
There is a strong drive for technologies that facilitate (i) the cost effective identification of proteins and their interactions with other proteins, as well as the role they play in metabolic, catabolic, and anabolic pathways; (ii) the cost effective profiling of proteins in terms of abundance and/or activity; (iii) the cost effective screening of massive numbers of small molecules and biologics against selected proteins.
Such technologies typically require a combination of speed, low reagent and sample consumption, multiplexing (i.e., analysis of multiple targets in parallel), low cost (particularly relating to any disposable elements), high assay repeatability, robust biochemical surfaces, and high sensitivity & selectivity. These requirements resulted in the development of protein microarrays which provide a means of mass producing surfaces, of typically a few centimeters square, comprising of a massive number of multiple target probes, usually proteins that are specific to, and bind to, known target proteins such as monoclonal antibodies (MABs). Much of this technology has evolved from gene microarrays. However, in contrast to gene arrays, where probes are typically synthetic oligonucleotides, protein microarrays suffer a number of important disadvantages: denaturation of complex protein structure due to either the protein attachment process and/or storage conditions; sensitivity and selectivity, due to the affinity and cross-reactivity of the binder protein used; and cost, due to the nature of the mass manufacturing technology used, often based on either silicon or a special glass. Furthermore, relative to the in situ synthesis of oligonucleotides, specific to target DNA/RNA, and used for the sequencing of DNA, identification of mutations (such as single nucleotide polymorphisms, SNPs), proteins cannot be built up in such a way. In situ synthesis of amino acids has been attempted but without any commercial success to date and needs to be added to a surface in a preformed fashion, i.e. as complete antibodies, mimics, or other form of binder protein. This necessarily limits the speed at which the process can be achieved, increases the costs, and requires access to such binder proteins.
In all cases, conventional approaches rely on a priori knowledge of target proteins and pathways in order to develop binder proteins, that form the basis of the biochemical probe arrays used to query those targets, and gain information on, for example, drug (small molecule, biologic) efficacy and toxicity.
The in situ synthesis of oligonucleotide arrays onto silicon substrates via the use of photo-labile groups and a series of masking and demasking steps, allowed Affymetrix to develop and produce its GeneChip™. This technology has provided a method of mass-producing such arrays on silicon using technology largely inherited from the semiconductor industry. These chips are, however, expensive, require lead times of up to one month, and provide oligomers of a limited number of bases. They also require a priori knowledge of the target genes. The length of the oligomers is limited by the photoactivation process used. It means that yield would be very poor for oligomers of greater than 25 bases in length. This significantly limits the sensitivity of this method for application such as gene-expression profiling, where only abundant genes are detected and not low copy numbers of genes, or in some cases their splice variants. This shortcoming can be circumvented by PCR amplification of the expressed RNA, however, artifacts are known to be created by such processes along with the fact that the biases introduced by PCR must be accounted for in the interpretation of results and gene expression profiling methodology used. The limited flexibility and high access costs associated with Affymetrix's technology have resulted in a number of companies, and users, producing glass-slide based arrays. This has been due in part to the increasing availability of so-called arrayers and spotters that, by various dispensing methods (e.g., ink-jets and pins) can deposit oligonucleotide or protein probe-containing reagents onto various substrates, and using a variety of surface chemistries and functional groups (e.g., amines and aldehydes, and the like) attach these probes to the surface. One method developed by Rosetta provides an efficient method of ink-jet printing nucloetide bases onto a substrate, which are subsequently in situ synthesized using conventional phosphoramidite chemistry, which does not suffer the aforementioned shortcomings of the Affymetrix approach.
Despite significant growth in the use of these microarray-based systems, limits in the applicability of these systems have led to dissatisfaction among users and many pharmaceutical companies have expressed interest in alternative methods of achieving high throughput screening.
Bead-based assays have therefore been developed which overcome the limitations of the microarray technology. The superior mixing in a bead-based array results in negligible mass transfer of target to bead, as opposed to microarrays where target diffusion is always mass-transfer limited. This results in faster time-to-result, reduced need for washing, and improved signal to noise ratios. Bead-based arrays also allow for greater spatial independence relative to microarrays, where probes occupy a fixed position on a substrate and cannot be individually manipulated. Such advantages are not only of interest to the tracking and manipulation of compounds in combinatorial libraries, but also to assays for application in diagnostics, prognostics, and drug discovery.
Luminex has developed a particle-based assay format employing micron-scale microspheres, whose coding is achieved through the mixing of two different fluorochromes (incorporated into polystyrene particles) in different molecular weight ratios. See, e.g., U.S. Pat. Nos. 6,268,222 and 5,736,330. Luminex has achieved 64 different codes by this method. A higher number of codes would require the use of 3 or more different fluorochromes. Spectral discrimination of codes becomes more challenging as do the costs associated with manufacturing the particles. Some coding schemes employ fluorescent spectra as a means of distinguishing particles. This can present a problem in media where background fluorescence occurs in the same frequency range as the coding. Such a situation would include the assay of various proteins in whole blood. Alternative approaches, currently under development, include that of Quantum Dot whose coded particles are distinguished by very narrow symmetric emission spectra, obtained by the nanometric tuning of semiconductor nanocrystals. See, e.g., U.S. Pat. No. 6,274,323. Also, SurroMed, discloses particles that are electroplated into the pores of an alumina membrane to which a silver electrode has been evaporated. See, e.g., WO 01/02374 and WO 00/65472. In the case of SurroMed, metals exhibiting different reflectivities are electro-deposited into these pores. The codes are provided by differential reflectivity. However, these technologies have limitations in practice, including the fact that attachment of proteins on semiconductor nanocrystals is non-trivial and tends to denaturation, and the utilization of metal substrates (such as by SurroMed) facilitates non-specific adsorption of non-target proteins, as well as limitations in their applicability such as the fact that presenting a reflectance-based code on a particle would be difficult to read in turbid media such as whole blood.
Finally, particle-based assay formats are typically run through flow cytometer instruments. Most of the above described particle based formats, however, require customized cytometers due to the need to detect optical emissions at different wavelengths to those that result solely from the binding of, for example, an antibody to an antigen (e.g. an antibiotic), and the subsequent attachment of a reporter antibody, to which is attached a fluorophore. Conventional sandwich immunoassay involves the washing of beads to which antigen and reporters have bound followed by excitation of the bead by a suitable wavelength source such that binding events could be detected, and in the case of a dose-response assay, quantitation of analyte measured at a specific point in time following exposure of antibody to antigen, through the relationship between emission intensity and analyte (target) concentration.